The mitotic spindle is a bipolar protein machine that uses microtubule (MT) based motors to coordinate chromosome segregation. We propose to evaluate the function of three MT-associated motor proteins, KRP130/KLP61F, ncd, and cytoplasmic dynein, with putative mitotic roles in Drosophila. Based on the ability of these motors to crosslink microtubules, we hypothesize that they function by mediating interactions between specifically oriented MTs. Moreover, we hypothesize that these motors have distinct but possibly overlapping functions which are likely to be reflected in spatial and temporal differences in their localization within the mitotic spindle. To test these hypotheses, we will pursue three specific aims. In the first, we will use high resolution immunomicroscopy in concert with detailed MT polarity analyses of Drosophila mitotic spindles to determine the localization of these motors during different stages of mitosis and the polarity orientation of the MTs with which they interact. In the second, we will investigate the MT-MT transport and bundling properties of the motors in vitro by combining purified motor holoenzymes with preparations of purified MTs. Particular attention will be given to the polarity relationships between crosslinked MTs. In the third, we will study mitotic spindle formation in cell-free extracts immunodepleted of various mitotic motors. We will then add back purified KRP130/KLP61F, ncd, and cytoplasmic dynein, individually or in combinations, to determine their ability to complement the resulting defects in spindle formation. Collectively, these studies will allow us to assign specific functions to individual motors and to determine how the functions of multiple motors are coordinated.